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Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in.

ABSTRACT: The CRISPR/Cas9 gene editing tool enables accessible and efficient modifications which (re)ignited molecular research in certain species. However, targeted integration of large DNA fragments using CRISPR/Cas9 can still be challenging in numerous models. To systematically compare CRISPR/Cas9's efficiency to classical homologous recombination (cHR) for insertion of large DNA fragments, we thoroughly performed and analyzed 221 experiments targeting 128 loci in mouse ES cells. Although both technologies proved efficient, CRISPR/Cas9 yielded significantly more positive clones as detected by overlapping PCRs. It also induced unexpected rearrangements around the targeted site, ultimately rendering CRISPR/Cas9 less efficient than cHR for the production of fully validated clones. These data show that CRISPR/Cas9-mediated recombination can induce complex long-range modifications at targeted loci, thus emphasizing the need for thorough characterization of any genetically modified material obtained through CRISPR-mediated gene editing before further functional studies or therapeutic use.

SUBMITTER: Rezza A

PROVIDER: S-EPMC6401152 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in.

Rezza Amélie A Jacquet Christelle C Le Pillouer Amélie A Lafarguette Florian F Ruptier Charlotte C Billandon Marion M Isnard Petit Patricia P Trouttet Séverine S Thiam Kader K Fraichard Alexandre A Chérifi Yacine Y

Scientific reports 20190305 1

The CRISPR/Cas9 gene editing tool enables accessible and efficient modifications which (re)ignited molecular research in certain species. However, targeted integration of large DNA fragments using CRISPR/Cas9 can still be challenging in numerous models. To systematically compare CRISPR/Cas9's efficiency to classical homologous recombination (cHR) for insertion of large DNA fragments, we thoroughly performed and analyzed 221 experiments targeting 128 loci in mouse ES cells. Although both technolo ...[more]

PMID: 30837594

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Project description:Gryllus bimaculatus is an emerging model organism in various fields of biology such as behavior, neurology, physiology and genetics. Recently, application of reverse genetics provides an opportunity of understanding the functional genomics and manipulating gene regulation networks with specific physiological response in G. bimaculatus. By using CRISPR/Cas9 system in G. bimaculatus, we present an efficient knockdown of Tyrosine hydroxylase (TH) and yellow-y, which are involved in insect melanin and catecholamine-biosynthesis pathway. As an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine, TH confines the first step reaction in the pathway. Yellow protein (dopachrome conversion enzyme, DCE) is also involved in the melanin biosynthetic pathway. The regulation system and molecular mechanism of melanin biogenesis in the pigmentation and their physiological functions in G. bimaculatus hasn't been well defined by far for lacking of in vivo models. Deletion and insertion of nucleotides in target sites of both TH and Yellow are detected in both F0 individuals and the inheritable F1 progenies. We confirm that TH and yellow-y are down-regulated in mutants by quantitative real-time PCR analysis. Compared with the control group, mutations of TH and yellow-y genes result in defects in pigmentation. Most F0 nymphs with mutations of TH gene die by the first instar, and the only adult had significant defects in the wings and legs. However, we could not get any homozygotes of TH mutants for all the F2 die by the first instar. Therefore, TH gene is very important for the growth and development of G. bimaculatus. When the yellow-y gene is knocked out, 71.43% of G. bimaculatus are light brown, with a slight mosaic on the abdomen. The yellow-y gene can be inherited stably through hybridization experiment with no obvious phenotype except lighter cuticular color. The present loss of function study indicates the essential roles of TH and yellow in pigmentation, and TH possesses profound and extensive effects of dopamine synthesis in embryonic development in G. bimaculatus.

Project description:Pierce's disease (PD) of grapevine (Vitis vinifera) is caused by the bacterium Xylella fastidiosa and is vectored by xylem sap-sucking insects, whereas Grapevine Red Blotch Virus (GRBV) causes Red Blotch Disease and is transmitted in the laboratory by alfalfa leafhopper Spissistilus festinus. The significance of anthocyanin accumulations in distinct tissues of grapevine by these pathogens is unknown, but vector feeding preferences and olfactory cues from host anthocyanins may be important for these disease etiologies. Phosphate, sugar, and UV light are known to regulate anthocyanin accumulation via miR828 and Trans-Acting Small-interfering locus4 (TAS4), specifically in grape by production of phased TAS4a/b/c small-interfering RNAs that are differentially expressed and target MYBA5/6/7 transcription factor transcripts for post-transcriptional slicing and antisense-mediated silencing. To generate materials that can critically test these genes' functions in PD and GRBV disease symptoms, we produced transgenic grape plants targeting TAS4b and MYBA7 using CRISPR/Cas9 technology. We obtained five MYBA7 lines all with bi-allelic editing events and no off-targets detected at genomic loci with homology to the guide sequence. We obtained two independent edited TAS4b lines; one bi-allelic, the other heterozygous while both had fortuitous evidences of bi-allelic TAS4a off-target editing events at the paralogous locus. No visible anthocyanin accumulation phenotypes were observed in regenerated plants, possibly due to the presence of genetically redundant TAS4c and MYBA5/6 loci or absence of inductive environmental stress conditions. The editing events encompass single base insertions and di/trinucleotide deletions of Vvi-TAS4a/b and Vvi-MYBA7 at expected positions 3 nt upstream from the guideRNA proximal adjacent motifs NGG. We also identified evidences of homologous recombinations of TAS4a with TAS4b at the TAS4a off-target in one of the TAS4b lines, resulting in a chimeric locus with a bi-allelic polymorphism, supporting independent recombination events in transgenic plants associated with apparent high Cas9 activities. The lack of obvious visible pigment phenotypes in edited plants precluded pathogen challenge tests of the role of anthocyanins in host PD and GRBV resistance/tolerance mechanisms. Nonetheless, we demonstrate successful genome-editing of non-coding RNA and MYB transcription factor loci which can serve future characterizations of the functions of TAS4a/b/c and MYBA7 in developmental, physiological, and environmental biotic/abiotic stress response pathways important for value-added nutraceutical synthesis and pathogen responses of winegrape.

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Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in.

Publications

Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in.

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